Functional and Structural Analysis of the Siderophore Synthetase AsbB through Reconstitution of the Petrobactin Biosynthetic Pathway from Bacillus anthracis

• Published in: The Journal of biological chemistry Vol. 287; no. 19; pp. 16058 - 16072
• Main Authors: Nusca, Tyler D., Kim, Youngchang, Maltseva, Natalia, Lee, Jung Yeop, Eschenfeldt, William, Stols, Lucy, Schofield, Michael M., Scaglione, Jamie B., Dixon, Shandee D., Oves-Costales, Daniel, Challis, Gregory L., Hanna, Philip C., Pfleger, Brian F., Joachimiak, Andrzej, Sherman, David H.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 04.05.2012; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Bacillus; Bacillus anthracis; Bacillus anthracis - genetics; Bacillus anthracis - metabolism; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Benzamides - chemistry; Benzamides - metabolism; Biocatalysis; Biosynthetic Pathways; Carbon-Nitrogen Ligases - chemistry; Carbon-Nitrogen Ligases - genetics; Carbon-Nitrogen Ligases - metabolism; Crystal Structure; Crystallography, X-Ray; Iron Acquisition; Iron Metabolism; Ligases - chemistry; Ligases - genetics; Ligases - metabolism; Models, Chemical; Models, Molecular; Molecular Sequence Data; Molecular Structure; Petrobactin; Polyamines - chemistry; Polyamines - metabolism; Protein Binding; Protein Structure and Folding; Protein Structure, Tertiary; Secondary Metabolism; Sequence Homology, Amino Acid; Siderophores; Siderophores - chemistry; Siderophores - metabolism; Substrate Specificity; Synthetase Structure; Crystal Structure; Siderophores; Bacillus anthracis; Iron Metabolism; Bacillus; Iron Acquisition; Synthetase Structure; Secondary Metabolism; Petrobactin
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M112.359349

Petrobactin, a mixed catechol-carboxylate siderophore, is required for full virulence of Bacillus anthracis, the causative agent of anthrax. The asbABCDEF operon encodes the biosynthetic machinery for this secondary metabolite. Here, we show that the function of five gene products encoded by the asb operon is necessary and sufficient for conversion of endogenous precursors to petrobactin using an in vitro system. In this pathway, the siderophore synthetase AsbB catalyzes formation of amide bonds crucial for petrobactin assembly through use of biosynthetic intermediates, as opposed to primary metabolites, as carboxylate donors. In solving the crystal structure of the B. anthracissiderophore biosynthesis protein B (AsbB), we disclose a three-dimensional model of a nonribosomal peptide synthetase-independent siderophore (NIS) synthetase. Structural characteristics provide new insight into how this bifunctional condensing enzyme can bind and adenylate multiple citrate-containing substrates followed by incorporation of both natural and unnatural polyamine nucleophiles. This activity enables formation of multiple end-stage products leading to final assembly of petrobactin. Subsequent enzymatic assays with the nonribosomal peptide synthetase-like AsbC, AsbD, and AsbE polypeptides show that the alternative products of AsbB are further converted to petrobactin, verifying previously proposed convergent routes to formation of this siderophore. These studies identify potential therapeutic targets to halt deadly infections caused by B. anthracis and other pathogenic bacteria and suggest new avenues for the chemoenzymatic synthesis of novel compounds. Background:asbABCDEF mediates petrobactin production and facilitates anthrax virulence. Results: Purified AsbA-E proteins reconstituted petrobactin assembly in vitro. The crystal structure and enzymatic studies of AsbB highlight its function and role in the siderophore pathway. Conclusion: AsbB characterization demonstrated reaction flexibility and substrate positions in the binding pocket. Significance: Siderophore synthetases represent promising antimicrobial targets, and characterization of these versatile enzymes enables creation of novel compounds.